**2. Possible different alternatives and approaches to the use of Cu**

## **2.1 Animal origins**

Chitoplant©, Enzicur© and other extracts from animal origin (*Lumbricus humus*, propolis, milk protein and hydrolyzed proteins) have been proposed to reduce downy mildew symptoms [12], as they can form semipermeable films protecting plant tissues and stimulating plant's defense mechanisms.

Chitosan hydrochloride is a kind of resistance promoter that enhances plant protection against pathogenic infections. It has proven effects against bacteria and fungi (such as *P. viticola*), and it was approved for use in agriculture as a plant protection product by European Commission6 [13].

However, their impacts on grapes and must quality have to be carefully assessed, as some studies point to negative effects. Garde-Cérdan et al. [14]. observed that both copper hydroxide and chitosan applications to the grapevines decreased the

<sup>3</sup> REGULATION (EU) 2018/1981 of 13 December 2018: total application of maximum 28 kg of Cu per hectare over a period of 7 years; Member States may in particular decide to set a maximum annual application rate not exceeding 4 kg/ha of Cu; expiration of approval: 31 December, 2025.

<sup>4</sup> https://www.demeter.net/wp-content/uploads/2021/04/20201204\_bfdi\_standard\_for2021\_final\_sc.pdf

<sup>5</sup> https://agriculture.gouv.fr/le-plan-ecophyto-quest-ce-que-cest

<sup>6</sup> Regulation (EU) number 563/2014 of 23 May 2014, following Regulation (EC) number 1107/2009 of the European Parliament and Council. https://eur-lex.europa.eu/legal-content/EN/TXT/PDF/?uri=CEL EX:32014R0563&from=EN

concentrations of all amino acids in must, except for Lys, and only when chitosan is applied alone. Romanazzi et al., [13], also recorded lower net photosynthesis, stomatal conductance, leaf area, and weight of leaves and pruned branches, as a consequence of chitosan treatments. The authors concluded that these side effects may be very risky for obtaining high berry quality.

Lactoperoxidase system (Enzicur©) is a natural anti-microbial system usually employed in the control of powdery mildew in various crops7 . The product is based on naturally occurring salts (potassium iodide and potassium thiocyanate) and the lactoperoxidase system, active in different animals including in the bovine liver. Enzymes (lactoperoxidase) and substrates. The LP-system is a non-immune defense system, that promotes the formation of reactive oxygen species that inactivate microorganisms by protein's peroxidation.

#### **2.2 Biocontrol agents (BCAs)**

*Bacillus subtilis* (Serenade Max©) and *Trichoderma harzianum* (Trichodex©) has been found as promising candidates for replacing Cu as a biocontrol agent for protecting against downy mildew [12], and other fungi diseases.

Among some tested antagonists, the highest efficiency was observed for *Trichoderma harzianum*-based products. Its efficiency was significantly higher in the treated plot when compared with untreated one but decreased just before harvest. However, this *Trichoderma harzianum*-based product did not provide a level of *P. viticola* control similar to Cu in some trials [15]. Despite the positive results found in some experimental studies, it was realized that the ability of *Thricoderma* (T39) to induce resistance depends on grapevine cultivars. Thus, it is necessary to understand which are the molecular components and signaling pathways modulating the response to this resistance inducer to apply this biocontrol to the most responsive cultivars, enhancing the benefits of this biocontrol treatment [16].

Other results [17] showed the relevance of environmental conditions on BCAs activity (four-year trial). Prevention of fungal sporous germination at least in some years could means an interaction between the pathogen and the microorganism that can lead to a reduction of severity of primary foci.

On other hand, *Bacillus* and *Trichoderma* strains have a great ability to produce a wide range of active molecules with broad effects on the control of different grapevine diseases, by preventively inducing plants systemic resistance or inhibiting other fungi diseases development.

These works show that microorganisms could be a promising tool to reach a reduction of primary inoculum and thus contribute to a low impact and sustainable agriculture.

#### **2.3 Cultural practices**

In the case of an epidemic disease like the downy mildew, combat strategies relied only on chemical control and its optimization. Sanitation measures targeting to reduce the overwintering inoculum and therefore, to reduce early and linearly the primary infection, and regulation of the crop load are a good management strategy [18].

Another relevant and additional strategy is the strict regulation of Cu spray rates. In the field, rates between 200 and 400 g Cu·ha−1 (equivalent to 5 and 10 mg Cu·m-<sup>2</sup> , respectively) was able to significantly reduce downy mildew (72–89% efficacy). These confirmed results (previously obtained from leaf disks assays in the lab), provided sufficient control, although it depends on the infection pressure [19].

<sup>7</sup> https://www.koppert.mx/enzicur/

*Alternatives to CU Applications in Viticulture: How R&D Projects Can Provide Applied… DOI: http://dx.doi.org/10.5772/intechopen.100500*

Forecasting models linked to Cu applications could be an interesting approach. **Coptimizer**<sup>8</sup> is a model-driven decision support system designed to help growers to optimize and track the use of Cu-based fungicides against grapevine downy mildew in European organic viticulture. Results showed that by using Coptimizer (including historical data and several experiments under field conditions), growers could be able to maintain the same level of protection applying only half the amount of the fungicide [20].

An innovative cultural practice has been recently tested consisting in the application of different cover crops mixtures to interfere with the dispersal of the soil-transient pathogen, such as *P. viticola* [21]. Fall sowing of cover crops allowed to have enough vegetation in spring, during the most relevant period of downy mildew primary infections, to delay the onset of first disease symptoms and reduce the final incidence of the epidemic. This cultural practice can result in a final saving in treatment numbers as well as a reduced amount of copper used during the first seasonal treatments.

In summary, when *P, viticola* pressure is low to intermediate, a reduction in the sprayed Cu quantity provides the same efficiency as standard strategies and allows to decrease two-fold to three-fold the sprayed Cu quantity [15].

#### **2.4 Inorganic materials**

Some inorganic salts have shown promising results under controlled conditions (greenhouse and potted plants) like potassium bicarbonates (Armicarb© and SaluKarb©); K-P product based on betaine, carbohydrates and amino acids (Gro-stim©); N-K products with oligosaccharide and glutathione (Kendal©) or Aluminum oxide and silicon oxide with S (Ulmasud©) showed to be as effective as Cu hydroxide treatment. However, in field trials, only the potassium bicarbonate (Armicarb©) provided control of infection on bunches greater than 60% [17].

#### **2.5 Microbial and plant product extracts or derivates**

Under controlled conditions (greenhouse and potted plants), some microbial extracts have shown a good efficacy to control downy mildew [17]. Extracts from inactivated *Pseudomonas aureofaciens* (Agat 25 K© and Diamant©) were an effective treatments at concentrations above 10%. This product was effective in field trials, providing control of infection on bunches greater than 60%.

Many plants' oils or water and alcohol extracts showed reduce downy mildew expression compared with the untreated control [12, 17], under controlled conditions (greenhouse and potted plants):


Therefore, plant and other extract products isolated used without Cu can reduce their efficacy when *P. viticola* cause a high pressure in the vineyards.

*Hedera helix* (leaves in water), *Quercus spec*. (bark in alcohol), *Primula veris* (roots in water), *Rhamnus frangula* (roots in alcohol), *Solidago spec*. (leaves in

<sup>8</sup> https://www.haifaup.co.il/startup/coptimizer

alcohol), *Salix spec*. (bark in water) showed promising effects in the laboratory [22], and these effects increased with the concentration of plant material used to obtain the extract. Extracts from *Rhamnus* and *Primula* had significant effects, reducing disease severity by 30–35% if applied after infection.

In field trials, some of the extracts, such as those from *Chenopodium quinoa*; *Inula viscosa*; *Melaleuca alternifolia* (Timorex©); *Salix alba*; *Solidago virgaurea and Salvia officinalis* provided more than 60% of control of bunches infection [17].

In general, preventive effects were much better in lab conditions (70–90% reduction of disease severity) than the results in field experiments (34–40% disease reduction) for the species tested [22]. In particular, *Yucca schidigera* (Norponin BS© liquid and Saponin©) has been also found as some of the most promising candidates for replacing Cu, because it provided more than 60% control of leaves and bunches infection [17]. However, some variability in *Yucca* extract efficiency under a low *P. viticola* pressure was already observed in some studies [15].

Trials with potted plants showed that *Salix* extract is a promising alternative to Cu, with no risk for the development of *P. viticola* resistant strains. Salix extract was as efficient, being the 4th day between elicitation and inoculation the appropriate moment to control the disease. Nevertheless, its action is strictly preventive and *Salix* extract should be applied before rainfall splash dispersion of fungi, which are impossible to forecast and in case of strong pressure this protection could be insufficient [23].

Therefore, available results also showed that the use of plant extracts (alone or in combinations among them) can reduce the doses of Cu and should be tested in future as a real alternative.

#### **2.6 Synthetic materials**

Under high *P. viticola* pressure, Cu-based treatments and potassium phosphonate (PP) are the most efficient products to control downy mildew. Beta-aminobutyric acid (BABA), benzothiadiazole, and high levels of polyoxyethylene sorbitan monooleate (Tween 80©) were as effective as the Cu hydroxide treatments in indoor trials [17], but no relevant effects were recorded in field trials.

Clay-based treatments such as Mycosin© are promising alternatives, giving in some trials a level of protection higher than 60% in leaves and bunches [15], but it is important to understand the impact of Al cations provided by this product. However, under a high disease pressure, the efficiency of these clay-based products is low for commercial vineyard protection.

Some vineyards trials in Germany and Austria showed that PP has a direct effect on *P. viticola*, and in addition, it activates the plant's defense mechanism (EFSA 20129 ) which is one of the basic principles of organic plant protection, as stated in the European Organic Regulation10. PP is absorbed by the plant and systemically distributed. Due to the distribution through the plant and the resistance-inducing effect, this substance particularly protects newly grown leaves and shoots. It also reaches the pathogens that have already penetrated the leaves. Apart from the protective effects, the substance also has a curative effect during the first days of infection and incubation (approx. 25% of elapsed incubation time).

PP was used in organic viticulture in a few countries as a plant strengthener until 2014. When used until the end of the flowering period, it showed great support of Cu products in protection against *P. viticola* under high infection pressure.

<sup>9</sup> https://efsa.onlinelibrary.wiley.com/doi/epdf/10.2903/j.efsa.2012.2963

<sup>10</sup> EU No. 834/2007

*Alternatives to CU Applications in Viticulture: How R&D Projects Can Provide Applied… DOI: http://dx.doi.org/10.5772/intechopen.100500*

Efficacy of PP, stone meal as well as new Cu formulations, has been recorded as good reference treatments (Folpan 80 WDG©, a.i. folpet© and "organic standard" mixture of Cu, sulfur and stone meal), when the *P. viticola*. The pressure was low, considering the low amount of total Cu applied (less than 2 kg/ha), the results were promising [24]. Moreover, the use of PP as a plant protection product in organic vineyards contributes to a Cu use reduction to levels <3 kg of pure Cu·ha−1·yr.−1, and it has been a practice adopted in Germany and Austria. Therefore, PP can be considered in Cu-reducing strategies.

However, PP were registered as plant protection agents in the EU and therefore, not listed or allowed to use in organic viticulture. This led to big problems in years with high infection pressure in different regions all over Europe (like in 2016).

#### **2.7 Other or new Cu formulations**

New Cu formulations available in the market showed efficacy similar to Cu hydroxide, however, are not efficient at low concentrations. Cu is a preventive fungicide allowed in organic agriculture that is active only in tissues where is applied (i.e. it is a non-systemic substance), so plant growth results in unprotected tissues. In areas where disease incidence is high, weekly Cu applications are made by growers increasing the risk of exceeding the fixed threshold.

Some low Cu formulations were able to control grape downy mildew in the field using a third (Glutex Cu 90©) or a sixth (Labicuper©) of the amount of Cu in comparison with the Cu hydroxide [25].

Cu gluconate (containing 8% of Cu2+) showed efficacy comparable to Cu hydroxide (containing 35% of Cu2+) in vineyard trials for managing downy mildew [26]. Acylbenzolar-s methyl (Bion 50 WG©) also confirmed its efficacy in vineyard trials.

Several new tested Cu formulations or mixtures provided effective disease control, but their efficacy levels decreased when lower rates of Cu2+ were used, and this pattern was similar for different formulations. Nevertheless, some general conclusions should be mentioned:


### **2.8 Technosoils or recovering soils. Measures to minimize the negative impacts of Cu in soils**

The use of amendments is a promising strategy for recovering soils. The use of limestone is an effective strategy to reduce Cu availability and phytotoxicity that has been used for many years [27–29]. Limestone promotes the increase in soil pH, causing deprotonation of acidic functional groups of reactive soil particles. This increases cation exchange capacity (CEC) and Cu adsorption, decreasing bioavailability and potential uptake by plants. Grapevines grown in soil treated with limestone showed increased growth, dry matter yield and photosynthetic efficiency in young grapevines in parallel with a lowest Cu concentration in root tissues.

Also, compost and biochar could help in slightly moderate acidic soils, with some positive effects of Cu2+ reductions by liming. In general, organic soil

amendments could achieve similar effects of Cu2+ reduction than liming, but they might be more valuable because of their beneficial effects on physicochemical soil characteristics and decreased risk of soil erosion. Therefore, compost and biochar are promising solutions because usually are non-expensive treatments and, biochar go beyond a simple liming effect [30].

Nevertheless, depending on its characteristics, the addition of organic amendments can result in the opposite effect (mobilization of Cu due to its complexation with low molecular weight and soluble organic compounds) [27]. Thus, the use of this agronomic practice must be evaluated case by case to not deteriorate the already altered soil conditions. In its turn, biochar can overcome this problem due to its different mechanisms of Cu complexation. Also, the application of treated coal fly ash can be a solution, especially if mixing with compost, overcoming the potential problems of Cu leaching and availability that may arise from the application of the compost alone.

Pyoverdine (Pvd) is a bacterial siderophore produced by some *Pseudomonas* species that can bind Cu in addition to iron in the soil. Pvd is expected to alter the dynamics and the ecotoxicity of Cu in vineyard soils. Cu phytoavailability depends to a great extent on Cu complexation in soil pore water, the latter being highly sensitive to pH: vineyard topsoils with pH ranging from 5.9 to 8.6 can present Cu mobility differences of six times and, a Cu phytoavailability differing by a factor of 5000 among them. The Pvd action depends on Fe soil availability, the soil composition (e.g. carbonate soils more easily mobilized Cu) and other factors [28].

Besides, many several bacterial strains can hyper-accumulate and/or sequestrate Cu [27].

Another example is the mutualistic association between arbuscular mycorrhizal fungi (AMF) and plant roots that can minimize the toxic effects of Cu in plants, due to the complexation of this element with organic substance produced and released by them. Also, AMF can store Cu in cellular compartments such as vesicles and spores [27].

#### **2.9 Modeling downy mildew**

In the last decades, many epidemiological models have been elaborated to better manage fungicide application schedules. The correlations among environmental factors, host susceptibility and the pathogen have been well known for a long time: the so-called 3–10 rule (3 days under 10 mm or more effective precipitation) was the first attempt to predict primary infections of *P. viticola* [31]. Similar models have been developed in France [32, 33], Germany [34], USA [35, 36], and Australia [37, 38]. Unfortunately, they often fail to predict the real development of epidemics and their practical use is restricted [39]. Empirical models have shown some critical restrictions and limitations being too simple, due to the lack of robust cause-effect relationships in many model equations and therefore, requiring some corrections and calibrations to adapt to grape-growing areas or environmental conditions different from those used for the model development [40].

A mechanistic dynamic model was recently elaborated in Italy [41], which accounts for the biological effects of weather on the different stages of the primary infection chain, from the progressive breaking of dormancy in the overwintering oospore population to infection establishment during the grapevine-growing season. The model of Rossi et al. [42] was evaluated in more than 100 vineyards in Italy (from 1995 to 2007) as well as in the environmental conditions in the province of Quebec, Eastern Canada, by comparing the time of first lesion occurrence predicted by the model with field observations [42, 43]. This model always showed very high accuracy [44] and when used to schedule fungicide application against

*Alternatives to CU Applications in Viticulture: How R&D Projects Can Provide Applied… DOI: http://dx.doi.org/10.5772/intechopen.100500*

downy mildew, allowed a reduction from 50 to 66% in fungicide applications, corresponding to an average saving of 174 and 224 €·ha−1, respectively [42]. Finally, it was integrated into a DSS named vite.net® [45].

Moreover, Caffi et al. [46] developed a weather-driven model to predict *P. viticola* population dynamics on grape leaf surfaces during a discrete wet period. The authors positively correlated the post-inoculation efficacy of two cooper fungicides with the proportion of *P. viticola* sporangia on a leaf that had not yet caused the infection. Model simulations suggested that the efficacy of a copper treatment increased when the environmental conditions were less conducive for disease development. Therefore, this model can be used to predict whether a fungicide application during a discrete infection period will be effective [42].

#### **2.10 Decision support system**

To help growers optimize the scheduling and dosages of fungicides against downy mildew, decision support systems were developed based on weather data, disease risk, and plant growth [45, 47].

The DSS vite.net® is an Internet-based platform for sustainable vineyard management [41] that has two main components: (i) an integrated system for real-time monitoring of vineyard data, and (ii) a web-based tool that analyses data by using mechanistic and, dynamic models that can predict grapevine growth, risk of disease infection, and residual protection by the last fungicide application. Each of these models has been published and their accuracy validated [45–50].

The combination of site-specific weather data, monitoring reports and advice from a DSS enables growers to protect their vineyards by modulating the frequency and timing of copper applications, based on disease risk [51].

The DSS vite.net® was tested in 21 organic farms and allowed the reduction of copper applications by an average of 24%, and the total amount of copper applied by 37% compared to a calendar-scheduling of copper application that provided the same level of protection in organic vineyards, with an average saving of 195 €·ha−1·year−1 compared to the common farm practice [52].

## **3. International legislation for PPPs application in vineyards**

Regarding the international legislation, the aim of reducing pesticides in viticulture has been addressed by European and international bodies and organizations.

The International Organization of Vine and Wine (OIV) is an intergovernmental organization established under the Agreement of 3 of April 2001, which is directly related to a previous agreement (OIV Treaty, 1924) made for the creation in Paris of an International Wine Office.

OIV is an intergovernmental organization (47 countries), comprising scientific and technical knowledge in grapevines, wine and wine-based beverages, table grapes, dried grapes, and other vine-based products, with an international reputation and generally recognized competencies. OIV countries represent more than 80% of total world wine production, and, being present in main continents worldwide.

The principal objective of OIV is to contribute to the international harmonization of existing practices and standards and, if needed, to draft new international standards for grapevine and wine products. OIV is also cooperating strongly with international organizations intergovernmental or non-intergovernmental like *Codex Alimentarius* or World Health Organization (WHO) among others.

Under a proposal from one of its group of experts (Vine protection and viticulture techniques "PROTEC"), OIV wanted to suggest some recommendations or good practices for minimizing the impacts associated with the application of plant protection products (PPPs) in vineyards.

A questionnaire was launched between 2014 and 2015 to its Member States and, answers showed some relevant results. For example, all of them have an Official List for prohibited and allowed products for grapevine protection and almost all of them (90%), has an official methodology about applications limits [53].

This new resolution (VITI 592–201811) includes some relevant points above described:


The resolution was completed with five annexes with most used models, decision support systems (DSS), conversion factors and an official list from departments and websites related to PPPs national rules and recommendations.

Talking about the EU framework12, some regulations should be considered, especially for organic production. As mentioned before, the rules for the implementation of organic production and labelling of organic products and control, describes quite well in article 5 and its Annex II. Pesticides — plant protection products, the use of Cu as fungicide up to 6 kg Cu per ha per year. For perennial crops, Member States may provide that the 6 kg Cu limit can be exceeded in a year provided that the average quantity actually used over 5 years consisting of that year and the four preceding years does not exceed 6 kg (it means 30 kg·ha−1 for 5 years

<sup>11</sup> https://www.oiv.int/public/medias/6450/oiv-viti-592-2018-en.pdf

<sup>12</sup> EC N° 834/2007

*Alternatives to CU Applications in Viticulture: How R&D Projects Can Provide Applied… DOI: http://dx.doi.org/10.5772/intechopen.100500*

limitation). Cu can be applied under the form of Cu hydroxide, Cu oxychloride, (tribasic), Cu sulphate, cuprous oxide, Cu octanoate.

Recently, this limit was revised (based on some EFSA reports) and consequently, Cu compounds were designated as candidate substances for substitution and reduced applications, restricting the use of plant protection products containing Cu compounds to a maximum application rate of 28 kg/ha of Cu over 7 years (i.e. on average 4 kg·ha−1·year−1). This is described in clause 15 in the first statement (EC N° 1981/2018) and it has two annexes with the use and forms of Cu and their specific provisions. This regulation shall be applied until 2025 or previous revision.

It also is remarked that Cu sulphate was authorized in organic wine production until 31 July 2015 (EC N° 203/2012).

Therefore, within this framework, the research focused on real alternatives to reduce or substitute the Cu products, with other active principles or compounds for controlling the pest and diseases in grapevines are a key challenge for the sustainability of the wine sector.
